Drug products and drug substances undergo forced degradation studies in which the stability of the drug substances and drug products are tested under extreme conditions to understand the degradation pathway of the active pharmaceutical ingredient (API), and to understand which impurities are degradation products and which are non-drug related. In such studies, mass balance is critical to understand degradation pathways and to ensure all impurities are accounted for. Mass balance is the practical application of the Law of Conservation of Mass, the total mass of the reactants consumed must equal the total mass of products formed, to chemical synthesis and degradation. With respect to drug stability and associated analyses, the International Conference on Harmonization (ICH) defines mass balance as “the process of adding together the assay value and levels of degradation products to see how closely these add up to 100% of the initial value, with due consideration of the margin of analytical error.” During a forced degradation study, impurities, specifically degradation products, must be summarized and measured relative to the API. When measuring the composition of the degraded sample using a chromatographic analysis system, the responsiveness of the detector of the analysis system to the API may be different than the responsiveness of the detector to the impurities. This can result in erroneous results for measurements of the concentrations of the impurities leading to corresponding errors in recovery for the mass balance.
One technique used to correct for differences in detector responsiveness to the API and to impurities involves generating calibration curves for the API and each impurity on the analytical system using samples with known concentrations of the API and of each impurity (e.g., known concentrations of standards). In a calibration curve, the slope of the detector response versus concentration is determined for each compound (e.g., for the API and for each impurity). This slope is referred to as the response factor for the compound. A relative response factor can be calculated for each impurity as the response factor of the impurity divided by the response factor of the API. This relative response factor can then be used to correct for the difference in detector responsiveness between the API and the impurities. Because the relative response factors are dependent on the specifics of the chromatographic method employed (e.g., column, gradient, mobile phases, etc.), the same conditions used for determining the RRF should be used for the subsequent mass balance analysis. Although this calibration curve-based technique for determining a relative response factor is well established and reliable, standards of known concentrations of the impurities are not always available. This calibration curve-based technique for determining relative response factors is also time-consuming as it requires measuring multiple different samples with known concentrations of the API and impurities to generate the calibration curves. Another technique of correcting for differences in detector responsiveness to the API and to impurities involves isolating the impurities from a sample and then using the impurities as standards when generating the calibration curves.
Nuclear magnetic resonance (NMR) may be used to determine relative response factors to correct for differences in detector responsiveness. NMR is nearly universal, quantitative on a molar basis, and well-established; however, it has numerous drawbacks (e.g., it is expensive and only detects compounds in relatively high concentrations). Further, it requires unique proton resonances and may not distinguish between closely related molecules.
Given the importance of stability studies for pharmaceuticals and accurate quantification of amounts of APIs and impurities, there is a need for a method of determining relative response factors for detection of substances in chromatographic separation that is not subject to the disadvantages described above.